Ketose 3-epimerase with improved thermal stability

Abstract

It is found that a variant enzyme with an improved thermal stability can be obtained by substituting a specific amino acid in the amino acid sequence of a ketose 3-epimerase originated from Arthrobacter globiformis and that D-psicose can be efficiently produced.

What is claimed is: 
     
       1. A variant of ketose 3-epimerase of SEQ ID NO: 1, said variant
 being selected from the group consisting of PM4, PM17, PM18, PM23, PM26, PM28, PM29, PM32, PM33, PM38, PM39, PM41, PM43, PM44, PM45, PM48, PM51, PM55, PM56, PM57, PM58, PM59, PM60, M61, PM62, PM63, PM64, and PM65, 
 wherein each variant has the following substitution(s) compared to the sequence of SEQ ID NO. 1: 
 
       
         
           
                 
                 
               
                     
                 
                   Ketose 
                     
                 
                   3-epimerase 
                   Substitutions in the Amino 
                 
                   variant 
                   Acid Sequence of SEQ ID NO: 1 
                 
                     
                 
                   PM4  
                   S137C and N173C 
                 
                   PM17 
                   H271C and D278C 
                 
                   PM18 
                   S67P, D68A, A69D and T70G 
                 
                   PM23 
                   R160A and H289X1 (X1 = VSARHKP (SEQ ID NO: 2)) 
                 
                   PM26 
                   R160A, H271C, D278C and H289X1 (X1 = VSARHKP 
                 
                     
                   (SEQ ID NO: 2)) 
                 
                   PM28 
                   S22T, S67P, D68A, A69D, T70G, R160A and H289X1 
                 
                     
                   (X1 = VSARHKP (SEQ ID NO: 2)) 
                 
                   PM29 
                   S22T, S67P, D68A, A69D, T70G, R160A, H177M and 
                 
                     
                   H289X2 (X2 = VSARHKP (SEQ ID NO: 2)) 
                 
                   PM32 
                   D278F 
                 
                   PM33 
                   A200L 
                 
                   PM38 
                   V237I 
                 
                   PM39 
                   A200K 
                 
                   PM41 
                   F275C and D278C 
                 
                   PM43 
                   H177M, A200K, V237I, F275C and D278C 
                 
                   PM44 
                   E75P 
                 
                   PM45 
                   T100P and D101V 
                 
                   PM48 
                   R160A and H289X2 (X2 = VSAR (SEQ ID NO: 3)) 
                 
                   PM51 
                   E75P, H177M and V237I 
                 
                   PM55 
                   R160A and H289X3 (X3 = VSARHK (SEQ ID NO: 4)) 
                 
                   PM56 
                   H6Y and H177M 
                 
                   PM57 
                   M110W and H177M 
                 
                   PM58 
                   S67P, A69D and T70G 
                 
                   PM59 
                   S22T and V34I 
                 
                   PM60 
                   D278L 
                 
                   PM61 
                   V237L 
                 
                   PM62 
                   V91I 
                 
                   PM63 
                   A95R 
                 
                   PM64 
                   S137K 
                 
                   PM65 
                   A270K. 
                 
                     
                 
             
                
                
                
                
                
               
               
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
       2. The variant of ketose 3-epimerase according to  claim 1 , which is immobilized on an immobilization carrier. 
     
     
       3. A method for preparing a D-ketose comprising:
 treating D-fructose with the variant of ketose 3-epimerase according to  claim 1 . 
 
     
     
       4. The variant of ketose 3-epimerase according to  claim 1 , wherein the variant exhibits
 (i) a ketose 3-epimerase enzyme activity at 50° C., higher than that of wild-type ketose 3-epimerase of SEQ ID NO: 1 measured under same condition, wherein the enzyme activity is measured after the variant and the wild-type ketose 3-epimerase are treated at 60° C. for 1 hour in 50 mM phosphate buffer of pH 8.0 and containing 2 mM magnesium sulfate, and/or 
 (ii) a ratio of the activity at 70° C. (t70) to that at 50° C. (t50), expressed as t70/t50, higher than that of wild-type ketose 3-epimerase of SEQ ID NO: 1.